The primary goal of this project is to use cryo-EM single particle analysis to obtain intermediate resolution structures (10-13 E) of the anthrax toxin protective antigen (PA) pore complex inserted into lipid nanodiscs. Accumulating PA pore nanodisc image data sets using cryo EM techniques will allow us to obtain much improved structural detail of the PA-pore -nanodisc complex. Normal mode flexible fitting procedures are being used with the prepore PDB to determine the extent of the conformational changes that accompany the PA prepore to PA pore transitions and during pH induced changes. Obtaining the intermediate resolution PA pore nanodisc structure will better define the critical internal pore interface involved in protein translocation. Specifically, we shall compare free PA pore nanodisc complexes with 1) GroEL bound PA pore nanodisc, 2) the PA F427A translocation mutant nanodisc and 3) the PA pore nanodisc-lethal factor full length or N terminal domain complex. This comparative structural approach will enable us to define structural elements involved in translocation control for projects 1 and 2. For the last project, we will be able to investigate a possible structural change in PA pore and possibly in PA pore-lethal factor complexes when the pH is decreased from 7 to 5.5, mimicking changes in binding interactions and transitions that occur during endosome acidification. Research and Biotechnology implications: The nanodisc-anthrax toxin pore complex may serve as a specific antigen delivery system for the active form of this toxin component. GroEL may be a universal tool to stabilize and facilitate membrane protein structure insertion into nanodiscs or other lipid structures thus aiding membrane protein structure determination. Our methods may serve as a basis model method for constructing and studying other bacterial toxin macromolecular complexes (e.g. Clostridia Toxin). Nanodisc complexes will enable us to probe and control kinetic and dynamics of binding interactions for large populations or single molecules. PUBLIC HEALTH RELEVANCE: Inhalation of the Bacillus anthracis spores, a potential bioweapon and subsequent production of exotoxin causes severe toxemia, bacteremia and death. We are implementing novel methods to isolate and view the structure of the anthrax toxin translocation pore complex (Protective Antigen) to apply this new structural knowledge toward developing new therapeutic anti-anthrax treatments and novel vaccines.